Process for Analyzing, for Separating, and for Isolating Individual Polar Protic Monomers and/or Oligomers

ABSTRACT

An improved process for separating and isolating individual polar protic monomer(s) and/or oligomer(s) on the basis of degree of polymerization. A liquid sample containing polar protic monomer(s) and/or oligomer(s) is introduced into a liquid chromatography (LC) column packed with a polar bonded stationary chromatographic phase. The individual polar protic monomer(s) and/or oligomer(s) are separated via a binary mobile phase elution. One or more individual fractions containing the monomer(s) and/or oligomer(s) are eluted. The polar protic monomer(s) and/or oligomer(s) may be proanthocyanidins, hydrolyzable tannins, oligosaccharides, oligonucleotides, peptides, acrylamides, polysorbates, polyketides, poloxarners, polyethylene glycols, polyoxyethylene alcohols or polyvinyl alcohols. The binary mobile phase comprises an A phase consisting essentially of a polar aprotic solvent and a B phase consisting essentially of a polar protic solvent. 
     A process for separating and isolating xanthine(s) (e.g., caffeine and theobromine) from polar protic monomer(s) and/or oligomer(s). A liquid sample containing xanthine(s) and polar protic monomer(s) and/or oligomer(s) is introduced into an LC column packed with a polar bonded stationary chromatographic phase. The xanthines are separated via an isocratic mobile phase elution, and one or more individual fractions containing the xanthines are eluted.

CROSS-REFERENCE TO RELATED APPLICATIONS

None.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to an improved process for separatingand preferably recovering individual polar protic monomers and/oroligomers, including without limitation flavan-3-ols, according to theirdegree of polymerization, using diol-phase liquid chromatography (LC).

It is known that individual flavan-3-ols exhibit distinct properties andhave distinct applications for human and animal use. Improved separationand recovery of individual flavan-3-ols on the basis of degree ofpolymerization will allow for more targeted and efficacious use thereof.

2. Discussion of the Related Art

Proanthocyanidins, the oligomers and polymers of flavan-3-ols, are thesecond most abundant natural plant phenolic after lignin. Theflavan-3-ol subunits are linked primarily through a carbon-carbon bondfrom the 4 position of one subunit to the 8 position of another subunit(C4→C8), and to a lesser extent through C4→C6 linkage.

Proanthocyanidins include B-type and A-type proanthocyanidins. In B-typeproanthocyanidins, the monomeric subunits are connected via interflavanlinkages of C4→C6 and/or C4→C8. Oligomers with exclusively C4→C8linkages are linear, while the presence of at least one C4→C6 bondresults in a branched oligomer. By contrast, A-type proanthocyanidinsare doubly linked oligomers containing linkages at C2-O-C7 as well as atC4→C6 or C4→C8.

The molecular weight of proanthocyanidins typically is expressed asdegree of polymerization (DP). Individual oligomers are commonlyreferred to as dimers, trimers, etc.

Procyanidins represent the largest class of proanthocyanidins. Gu et al.showed that out of 41 foods found to contain proanthocyanidins, 27contained procyanidins. (J. Agric. and Food Chem. 51 (2003) 7513).Procyanidins may consist of (−)-epicatechin, (+)-epicatechin,(+)-catechin and/or (−)-catechin subunits, as well as gallated catechinssuch as (−)-catechin gallate, (+)-catechin gallate, (−) epicatechingallate and/or (+)-epicatechin gallate.

It is known that proanthocyanidins play important roles in colorstability, astringency and bitterness in plant foods. (See, e.g.,Haslam, “Practical Polyphenols: From Molecular Recognition andPhysiological Action” (Cambridge U. Press, 1998)). However, thenotoriety of proanthocyanidins has increased due to the potential healthbenefits of these phenolic compounds. (See, e.g., Bagchi et al.,Toxicology 148 (2000) 187; Foo et al., J. Natural Products 63 (2000)1225; Steinberg et al., Am. J. Clin. Nutri. 77 (2003) 1466).

It is also known that individual procyanidin oligomers present specificcharacteristics and potential benefits for use in humans and animals.For example, Tempesta discloses that procyanidin oligomers having adegree of polymerization (DP) of 2-11 possess significant antiviralactivity, and are useful in treating warm-blooded animals, includinghumans, infected with paramyxovaridae such as respiratory syncytialvirus, orthomyxovaridae such as influenza A, B and C, and herpes virusessuch as Herpes Simplex virus. (U.S. Pat. No. 5,211,944). Romanczyk Jr.,et al. disclose antineoplastic compositions comprising procyanidinoligomers having a DP of 3-11 together with a suitable carrier. (U.S.Pat. No. 5,554,645). Romanczyk, Jr. et al. also disclose thatprocyanidin oligomers having a DP of 5-12 are useful as antioxidants.(U.S. Pat. No. 5,891,905). Schmitz et al. disclose the use of cocoaprocyanidin oligomers (DP of 2-18) together with acetylsalicylic acid asanti-platelet therapy. (U.S. Pat. No. 6,524,630).

Given their structural complexity and diversity in nature, the historyof proanthocyanidin analysis is rich. (Santos-Buelga et al., in“Processes in Polyphenol Analysis,” Royal Society of Chemistry,Cambridge, 2003, p. 267). Lea described the use of normal-phase highperformance liquid chromatography (NP-HPLC) for procyanidin analysis (J.Sci. Food and Agriculture 30 (1979) 833), and also observed that using aSephadex LH-20 column under isocratic conditions resulted in an elutionorder where the larger oligomers were retained longer than the smalleroligomers. (Lea et al., Am. J. Enology and Viticulture 30 (1979) 289).Wilson et al. disclosed using a gradient mobile phase in connection withtetrahydrofuran-hexane-acetic/formic acid-isopropanol over a cyanocolumn to achieve partial separation based on DP of apple juiceprocyanidins. (Sci. Food. Agric. 32 (1981) 257).

Significant improvements in the separation and resolution of procyanidinoligomers have been achieved on silica stationary phases. (See, Rigaudet al., Chromatogr. 654 (1993) 179; Cheynier et al., Processes inEnzymology 299 (1999); Natsume et al., Biosci. Biotechnol. Biochem. 64(2000) 2581). Resolution of procyanidin oligomers up to the pentamer(DP=5) has been obtained. Hammerstone et al. disclosed modifications ofthis process leading to improvements in resolution of monomers throughthe decamers in the analysis of unfermented, defatted cacao beans. (J.Agric. and Food Chem. 47 (1999) 490). Gu et al. disclosed still furtherimprovements leading to the elution of a decamer (DP>10), as well asenhancement in overall peak shape and resolution. (J. Agric. and FoodChem. 50 (2002) 4852).

These HPLC processes use gradient mobile phases consisting of methylenechloride-methanol-acetic/formic acid-water to achieve separation ofprocyanidin oligomers out to the pentamer with an alternate process forseparation of apple procyanidins using hexane-methanol-ethyl acetate andhexane-acetone over a silica column. (Yanagida et al., J. Chromatogr. A890 (2000) 251).

However, current HPLC processes, including those of Gu et al., haveseveral shortcomings. First, the use of chlorinated solvents such asmethylene chloride (also referred to as dichloromethane) presents safetyconcerns. This is especially an issue when isolated fractions from thelarger scale systems may be targeted for further biological study. It isknown that exposure to methylene chloride affects the skin, eyes,central nervous system (CNS), and cardiovascular system, and thatshort-term exposure can cause fatigue, weakness, sleepiness,light-headedness, numbness of limbs, tingling skin, nausea, andirritated skin and eyes. Chronic exposure to methylene chloride has beenlinked to cancer of the lungs, liver, and pancreas in laboratoryanimals. Methylene chloride also is a mutagen that may cause birthdefects if women are exposed to it during pregnancy.

Tetrahydrofuran (THF), another common HPLC solvent, is known to irritatethe eyes of human subjects, as well as the mucous membranes and thegastrointestinal tract. Overexposure to THF may cause coughing,shortness of breath, dizziness, central nervous system (CNS) depression,intoxication and collapse.

Other problems with current HPLC processes for separating procyanidinoligomers include the historical problem associated with normal phase(NP) separation of procyanidins when using silica as the stationaryphase, viz., column to column variability. Frequently, oligomers havereduced peak intensities or are not detected at all, as they are thoughtto be adsorbed on the silica surface. The use of water in the mobilephase—as required for peak shape in the NP separation ofprocyanidins—further degrades column to column reproducibility. Also,from a practical standpoint, most current HPLC processes for separatingindividual flavan-3-ols, including procyanidin oligomers, involve theuse of tertiary or quaternary mobile phases, and thus are beyond thecapabilities of analytical laboratories lacking sophisticated quaternaryHPLC pumps.

What is needed is an improved process for identifying the individualpolar protic oligomers on the basis of DP, as well as a process forseparating and recovering individual polar protic oligomers based on DPthat avoids use of dangerous and environmentally hazardous solvents,that provides improved separation and recovery of individual polarprotic oligomers, that is suitable for use in analytical laboratoriesequipped only with a customary binary HPLC pump, and that is suitablefor the recovery of specific oligomers on a preparative scale.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an improved process for separating andeluting individual polar protic monomer(s) and/or oligomer(s). Theprocess comprises the steps of: (i) introducing a liquid samplecontaining the monomer(s) and/or oligomer(s) into a liquidchromatography (LC) column packed with a polar bonded stationary phase;(ii) separating the individual monomer(s) and/or oligomer(s), on thebasis of degree of polymerization, by passing a binary mobile phasecomprising an A phase consisting essentially of a polar aprotic solventand a B phase consisting essentially of a polar protic solvent throughthe column; and (iii) eluting one or more individual fractionscontaining the monomer(s) and/or oligomer(s). In certain preferredembodiments, one or more of the eluted individual monomer(s) and/oroligomer(s) may be recovered for use in a food product, a medicinal foodproduct, a nutraceutical, or a pharmaceutical product, by drying orother conventional means. The eluted individual monomer(s) and/oroligomer(s) also may recovered for use as standards in otherseparations.

The polar protic monomer(s) and/or oligomer(s) may be proanthocyanidins,hydrolyzable tannins, oligosaccharides, oligonucleotides, peptides,acrylamides, polysorbates, polyketides, poloxamers, polyethyleneglycols, polyoxyethylene alcohols or polyvinyl alcohols. Where the polarprotic monomer(s) and/or oligomer(s) are proanthocyanidins, they may beproapigeninidins, proluteolinidins, protricetinidins, propelargonidins,procyanidins, prodelphinidins, proguibourtinidins, profisetinidins,prorobinetinidins, proteracacinidins and/or promelacacinidins.Preferably, the monomer(s) are epicatechin and/or catechin, and theoligomer(s) are procyanidin oligomers thereof. Also preferably, thesample containing the monomer(s) and/or oligomer(s) is a polar, defattedcocoa extract.

The amount of extract present in the liquid sample introduced into theLC column preferably is greater than 10 milligrams. More preferably, theamount of extract is greater than 100 milligrams. Even more preferably,the amount of extract is greater than 1 gram.

The polar bonded stationary phase may be a diol phase, a glycerol phase,an amino phase, a cyano phase, a trimethylsilyl phase, a dimethylsilylphase, a propyl phase, a butyl phase, a pentyl phase, a hexyl phase, aphenyl phase, a halogenated phase and a nitro phase. Preferably, thestationary phase is a diol phase or a glycerol phase.

In the binary mobile phase, which preferably is aqueous, the polaraprotic solvent may be any of acetonitrile, acetone, cyclohexanone,methyl ethyl ketone, methyl tert-butyl ether, diethyl ether, dimethylether, methyl acetate, ethyl acetate and nitromethane, and the polarprotic solvent may be any of methanol, ethanol, n-propanol, isopropanol,n-butanol and isobutanol.

The proportion of polar aprotic solvent in the A phase may be up to 100percent by volume, and the proportion of the polar protic solvent in theB phase may be up to 100 percent by volume. The remainder of the A phaseand B phase may be any mineral or organic acid and/or water. Preferably,the proportion of polar aprotic solvent in the A phase and polar proticsolvent in the B phase is greater than 50 percent by volume. Even morepreferably, the proportion of polar aprotic solvent (A phase) and polarprotic solvent (B phase) is greater than 90 percent by volume. In apreferred embodiment, the polar aprotic solvent in the A phase is about98% acetonitrile by volume, and the polar protic solvent in the B phaseis about 95% methanol by volume.

The polar bonded stationary phase preferably has a particle size fromabout 3 μm to about 10 μm. The LC column preferably has a diameter of atleast ten (10) millimeters.

The present invention also provides a system for separating and elutingindividual polar protic monomer(s) and/or oligomer(s) on the basis ofdegree of polymerization. The system comprises an LC column packed witha polar bonded stationary chromatographic phase; and a binary mobilephase comprising an A phase consisting essentially of a polar aproticsolvent and a B phase consisting essentially of a polar protic solventfor eluting one or more individual fractions containing the monomer(s)and/or oligomer(s).

The polar bonded stationary phase may be any of a diol phase, a glycerolphase, an amino phase, a cyano phase, a trimethylsilyl phase, adimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, ahexyl phase, a phenyl phase, a halogenated phase and a nitro phase.Preferably, the polar bonded stationary chromatographic phase is a diolphase or a glycerol phase. Also preferably, the polar bonded stationarychromatographic phase has a particle size from about 3 μm to about 10μm. The column preferably has a diameter of at least ten (10)millimeters.

In the binary phase, the polar aprotic solvent in the A phase may be anyof acetonitrile, acetone, cyclohexanone, methyl ethyl ketone, methyltert-butyl ether, diethyl ether, dimethyl ether, methyl acetate, ethylacetate and nitromethane. The polar protic solvent in the B phase may beany of methanol, ethanol, n-propanol, isopropanol, n-butanol andisobutanol.

Suitable polar protic monomer(s) and/or oligomer(s) for the systeminclude proanthocyanidins, hydrolyzable tannins, oligosaccharides,oligonucleotides, peptides, acrylamides, polysorbates, polyketides,poloxamers, polyethylene glycols, polyoxyethylene alcohols and polyvinylalcohols. Where the polar protic monomer(s) and/or oligomer(s) areproanthocyanidins, they may be proapigeninidins, proluteolinidins,protricetinidins, propelargonidins, prodelphinidins, proguibourtinidins,profisetinidins, prorobinetindins, proteracacinidins and/orpromelacacinidins. In a preferred embodiment, the monomer(s) areepicatechin and/or catechin, and the oligomer(s) are procyanidinoligomers thereof.

The proportion of polar aprotic solvent in the A phase may be up to 100percent by volume, and the proportion of the polar protic solvent in theB phase may be up to percent by volume. The remainder of the A phase andB phase may be any mineral or organic acid and/or water. Preferably, theproportion of polar aprotic solvent in the A phase and polar proticsolvent in the B phase is greater than 50% by volume. Even morepreferably, the proportion of polar aprotic solvent (A phase) and polarprotic solvent (B phase) is greater than 90% by volume. In a preferredembodiment, the polar aprotic solvent in the A phase is about 98%acetonitrile by volume, and the polar protic solvent in the B phase isabout 95% methanol by volume.

The present invention also provides a process for separating andisolating xanthine(s) from polar protic monomer(s) and/or oligomer(s).The process comprises the steps of: (i) introducing a liquid samplecontaining the xanthine(s) and polar protic monomer(s) and/oroligomer(s) into a liquid chromatography (LC) column packed with a polarbonded stationary chromatographic phase; (ii) separating the xanthine(s)from the monomer(s) and/or oligomer(s) by passing an isocratic mobilephase consisting essentially of a polar aprotic solvent through thecolumn; and (iii) eluting one or more individual fractions containingthe xanthine(s). The xanthine(s) preferably are caffeine and/ortheobromine. In a preferred embodiment, the liquid sample is a polar,defatted cocoa extract. In certain preferred embodiments, the elutedindividual xanthine(s) may be recovered for use in a food product, amedicinal food product, a nutraceutical, or a pharmaceutical product, bydrying or other conventional means. The eluted individual xanthine(s)also may be recovered for use as standards in other separations.

The polar bonded stationary phase may be any of a diol phase, a glycerolphase, an amino phase, a cyano phase, a trimethylsilyl phase, adimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, ahexyl phase, a phenyl phase, a halogenated phase and a nitro phase.Preferably, the stationary phase is a diol phase or a glycerol phase.

In the isocratic mobile phase, which preferably is aqueous, the polaraprotic solvent preferably is acetonitrile.

The proportion of polar aprotic solvent in the isocratic mobile phasepreferably is at least 90 percent by volume. The remainder of the mobilephase may be any mineral or organic acid and/or water. In a preferredembodiment, the polar aprotic solvent in the isocratic mobile phase isabout 99% acetonitrile by volume.

The present invention also provides a system for separating andisolating xanthine(s) from polar protic monomer(s) and/or oligomer(s).The system comprises a liquid chromatography column packed with a polarbonded stationary chromatographic phase; and an isocratic mobile phaseconsisting essentially of a polar aprotic solvent for eluting one ormore individual fractions containing the xanthine(s).

The polar bonded stationary phase may be any of a diol phase, a glycerolphase, an amino phase, a cyano phase, a trimethylsilyl phase, adimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, ahexyl phase, a phenyl phase, a halogenated phase and a nitro phase.Preferably, the stationary phase is a diol phase or a glycerol phase.Also preferably, the stationary phase has a particle size from about 3μm to about 10 μm. The column preferably has a diameter of at least ten(10) millimeters.

In the isocratic mobile phase, the polar aprotic solvent preferably isacetonitrile. The proportion of polar aprotic solvent in the isocraticmobile phase preferably is greater than 90% by volume. The remainder maybe any mineral or organic acid and/or water. In a preferred embodiment,the polar aprotic solvent in the isocratic mobile phase is about 99% byvolume acetonitrile.

The present invention also provides a process for separating and elutingxanthine(s) and individual polar protic monomer(s) and/or oligomer(s).The process comprises the steps of: (i) introducing a liquid samplecontaining the xanthine(s) and polar protic monomer(s) and/oroligomer(s) into a liquid chromatography column packed with a polarbonded stationary chromatographic phase; (ii) separating the xanthine(s)from the monomer(s) and/or oligomer(s) by passing an isocratic mobilephase consisting essentially of a polar aprotic solvent through thecolumn, and eluting one or more individual fractions containing thexanthine(s); (iii) subsequently separating the individual monomer(s)and/or oligomer(s), on the basis of degree of polymerization, by passinga binary mobile phase comprising an A phase consisting essentially of apolar aprotic solvent and a B phase consisting essentially of a polarprotic solvent through the column; and (iv) eluting one or moreindividual fractions containing the monomer(s) and/or oligomer(s). Incertain preferred embodiments, the eluted xanthine(s) and/or one or moreof the eluted individual monomer(s) and/or oligomer(s) may be recoveredfor use in a food product, a medicinal food product, a nutraceutical, ora pharmaceutical product, by drying or other conventional means. Theeluted xanthine(s) and/or eluted individual monomer(s) and/oroligomer(s) also may be recovered for use as standards in otherseparations.

The xanthine(s) preferably are caffeine and/or theobromine. Themonomer(s) and/or oligomer(s) may be any of proanthocyanidins,hydrolyzable tannins, oligosaccharides, oligonucleotides, peptides,acrylamides, polysorbates, polyketides, poloxamers, polyethyleneglycols, polyoxyethylene alcohols and polyvinyl alcohols. The polarbonded stationary phase may be any of a diol phase, a glycerol phase, anamino phase, a cyano phase, a trimethylsilyl phase, a dimethylsilylphase, a propyl phase, a butyl phase, a pentyl phase, a hexyl phase, aphenyl phase, a halogenated phase and a nitro phase.

These and other objects and embodiments are disclosed or will be obviousfrom the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of monomeric flavan-3-ols and procyanidin oligomerslinked through carbon-carbon C4→C8 and C4→C6 linkage.

FIG. 2. Diol-phase HPLC fluorescence trace of procyanidins fromunfermented cacao beans.

FIG. 3. Diol column HPLC trace of separation of polar protic monomersand oligomers from Cinnamomum loureirii acetone buffer extract.

FIG. 4. HPLC chromatograms of procyanidins, using Lichrosphere Sihca(bottom) and Develosil Diol (top) as stationary phase.

FIG. 5. Graph of percentage increase in peak area of diol-versus-silicastationary phase in HPLC for procyanidin oligomers, by degree ofpolymerization.

FIG. 6. Preparative-phase HPLC profile of CP extract.

FIG. 7. FLD traces of individual fractions collected from HPLCseparation of CP extract for oligomeric fractions DP=1 through 7.

FIG. 8. LC trace chromatogram of preparative system for separatingcaffeine and theobromine from CP extract.

FIG. 9. HPLC analytical chromatograms of isolated fractions fromsemi-preparative diol separation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses an improved process for separating andisolating polar protic monomer(s) and/or oligomer(s) on the basis ofdegree of polymerization, using a diol-based LC system having a binarymobile phase to elute one or more individual fractions containingindividual polar protic monomer(s) and/or oligomer(s), as well as asystem for same.

FIG. 1 shows the structure of monomeric flavan-3-ols and procyanidinoligomers linked through C4→C8 and C4→C6 linkage.

The improved processes and system of the present invention, using adiol-based stationary phase LC column and binary mobile phase comprisingan A phase of acetonitrile and acetic acid (CH₃CN:HOAc) in a 98:2 (v/v)mix, and a B phase of methanol, water and acetic acid (CH₃OH:H₂O:HOAc)in a 95:3:2 (v/v/v) mix, overcome the problem of provide for betterseparation of individual oligomers, including especially higher orderoligomers. FIG. 4, shows a comparison of chromatograms using diol (top)and silica (bottom) as the stationary phase. As shown, the diolstationary phase provided for greater separation of individualoligomers, and in particular provided from greater separation ofoligomers having DP of 7 or higher. It will be appreciated that theimproved processes of the present invention also are more useful thancurrent processes, in that they can be used with a wider range ofsolvents (including water), and can be used in laboratories equippedonly with a binary LC pump. Other benefits inherent in the improvedprocesses include the ability to analyze compounds (i.e., otherflavenoids, caffeine, theobromine) in addition to procyanidins in asingle LC run, especially in conjunction with mass spectrometry (LC-MS).A process for separating caffeine and theobromine, employing amodification of the improved process disclosed and claimed, is set forthherein.

The processes and system are more fully described below. While thespecifically disclosed processes and system involve cocoa procyanidins,it will be understood by those of ordinary skill in the art that thedisclosed processes and system are suitable for separating any polarprotic monomer(s) and/or oligomer(s), including without limitationproanthocyanidins (i.e., proapigeninidins, proluteolinidins,protricetinidins, propelargonidins, prodelphinidins, proguibourtinidins,profisetinidins, prorobinetindins, proteracacinidins andpromelacacinidins), hydrolyzable tannins, oligosaccharides,oligonucleotides, peptides, acrylamides, polysorbates, polyketides,poloxamers, polyethylene glycols, polyoxyethylene alcohols and polyvinylalcohols. Substances containing polar protic monomer(s) and/oroligomer(s), in addition to cocoa extract, include peanut skins,cinnamon, blueberries, apples, sorghum, hawthorne, cranberries andgrapes. In addition, the disclosed processes and system are suitable forseparating other sugar polymers, such as maltosaccharides,cyclodextrins, N-acetylchitooligosaccharides and pridylamino sugarchains.

All solvents (methylene chloride, acetonitrile, methanol, acetic acid)were chromatographic grade and purchased from Fisher Scientific(Fairlawn, N.J.). Ethanol was USP-food grade purchased fromSigma-Aldrich (Milwaukee, Wis.). Water was de-ionized using aMilli-Q-Water Purification System from Millipore (Bedford, Mass.).

Sample preparation: CP extract. A cocoa polyphenol (CP) extract wasprepared via a multi-step process aimed at minimizing degradation of thecocoa procyanidins. Cocoa beans were harvested, washed free of pulp anddried. Under ambient conditions the dried beans were expeller-pressed toremove cocoa butter. The expeller cake was then ground and extractedwith ethanol:water (70:30 v/v). Solids were removed by centrifugation.The extract liquid was evaporated under reduced pressure to remove theethanol and finally spray dried.

Sample preparation: CP extract from cacao seeds. Fresh unfermented cacaoseeds (30 g) were freeze-dried (16.9 g). Freeze-dried seeds (10.2 g)were defatted with hexane, and a sub-sample of the defatted beans (6.3g) was milled and extracted thrice with 40 mL acetone:water:acetic acid(70:29.5:0.5, v/v/v) while sonicating (10 min, 50° C). Acetone wasremoved from the combined extractions by rotary evaporation underreduced pressure. The remaining liquid was freeze dried to afford ared-purple residue (1.13 g).

Normal-phase high performance liquid chromatogaphy mass spectrometry(NP-HPLC-MS) analysis of CP extracts and purified oligomers. Separationand characterization of procyanidin oligomers in cocoa extracts andprocyanidin oligomeric fractions obtained from the preparative systemdescribed below was performed by NP-HPLC-MS. Separations were conductedon an Agilent 1100 HPLC system equipped with an autosampler, quaternaryHPLC pump, column heater, diode array detector, and fluorescencedetector. The HPLC was interfaced to an HP Series 1100 mass selectivedetector (Model G1946A) equipped with an API-ES ionization chamber. Thecolumn used was Develosil Diol (250×4.6 mm I.D., 5μ particle size)purchased from Phenomenex (Torrance, Calif.). The binary mobile phaseconsisted of (A) acetonitrile:acetic acid (98:2, v/v) and (B)methanol:water:acetic acid (95:3:2, v/v/v). Separations were effected bya linear gradient (of the mobile phase) at 30° C. with an 0.8 mL/minflow rate as follows: 0-35 minutes, 040% B; 35-40 min, 40% B isocratic;4045 min, 40-0% B, followed by a 5 minute re-equilibrate time. Eluentwas monitored by fluorescence detection (FLD) (excitation wavelength=276nm, emission wavelength=316 nm). Extracts and purified fractions werecharacterized by MS processes and parameters adapted from Hammerstone etal., J. Agric. and Food Chem. 47 (1999) 490, which are incorporatedherein by reference. Ionization reagents were added via a tee in theeluant stream of the LC just prior to the mass spectrometer anddelivered via an LC pump. Conditions for analysis in the positive ionmode included introduction of 0.05M NaCl at a flow rate of 0.05 mL/minto assist ionization, a capillary voltage of 3.5 kV, a fragmentorvoltage of 100 V, a nebulizing pressure of 25 psig, and a drying gastemperature of 350° C. Conditions for analysis in the negative ion modeincluded 1.5 M NH₄OH as a buffering agent at a flow rate of 0.09 mL/minfor 29 minutes, and then at 0.05 mL/min. Capillary voltage was 3 kV,fragmentor voltage was 75 V, nebulizing pressure was 25 psig and dryinggas temperature was 350° C.

Use of ammonium hydroxide was omitted from the MS analysis. Samples weredissolved in acetone:water:acetic acid (70:29.5:0.5, v/v/v) or mobilephase and filtered through 0.45 μm PTFE syringe filters prior toinjection.

Comparative NP-HPLC of CP extracts conducted on both Lichrosphere silicaand Develosil diol stationary phases. Separations of CP fractions wereconducted on both Lichrosphere Silica and Develosil Diol under thechromatographic conditions described by Adamson et al., J. Agric. andFood Chem. 47 (1999) 4184. The chromatographic system was an Agilent1100 Series HPLC system equipped with a temperature-controlledautosampler, quaternary pump, column heater, and fluorescence detector.The columns used were Lichrosphere Silica (250×4.6 mm, 5μ, 100 Å poresize) and Develosil Diol. The chromatographic mobile phase consisted ofmethylene chloride (CH₂Cl₂), methanol (CH₃OH), and acetic acid:water(1:1) (HOAc:H₂O). Starting mobile phase conditions were 82% CH₂Cl₂, 14%CH₃OH and 4% (HOAc:H₂O). Subsequently CH₃OH was ramped to 28.4% after 30minutes, 42.8% after 50 minutes and 86.0% after 51 minutes. Throughoutthe chromatographic run, the HOAc:H₂O ratio was held at a constant 4%.Fluorescence detection was conducted with an excitation wavelength of276 nm and emission at 316 nm. All samples were prepared by dissolutionin acetone:water:acetic acid (70:29.5:0.5) and then filtered through0.45 μm PTFE syringe filters for subsequent HPLC injection.

Preparative NP-HPLC of CP extracts. An Agilent 1100 series preparativepump was connected to a HP 1050 UV detector and a Kipp and Zonenrecorder. Preparative separation of procyanidins was achieved using aDevelosil Diol (300×50 mm I. D., 100 μm particle size) column purchasedfrom Phenomenex (Torrance, Calif.). The mobile phase consisted ofsolvents A (CH₃CN:HOAc, 99:1 v/v) and B (CH₃OH:H₂O:HOAc, 95:4:1 v/v/v)using a linear gradient of 0-30% B for 35 min. followed by isocraticperiod for 30 min, finally increasing to 80% B. The re-equilibrationtime was 10 min. The flow rate was set to 55 ml/min, the columntemperature was room temperature (23+2° C.). The detector was set to 280nm. Column loading was performed with a manual sample injector RheodyneValve model 7725 equipped with a 2 mL injection loop. CP extract wasdissolved in mobile phase, centrifuged and filtered through 0.45 μm PTFEsyringe filter prior to injecting.

Semi-preparative separation of procyanidins was also achieved using aDevelosil Diol (250×21.5 mm I. D., 100 μm particle size) columnpurchased from Phenomenex (Torrance, Calif.). The mobile phase consistedof the same composition as discussed above. The linear gradient was0-30% B for 45 min followed by isocratic period for 20 min, finallyincreasing to 85% B to wash any remaining residues off the column. Theflow rate for this column was 15 mL/min.

FIG. 2 shows the chromatogram of unfermented, defatted cacao seedextract. The labels 2-14 indicate the degree of polymerization ofprocyanidins in the peaks. Compounds were eluted according to theirdegree of polymerization and were characterized by LC-MS as flavan-3-olmonomers and oligomers up to the tetradecamer (DP=14). As disclosed inGu et al., the flavan-3-ol monomeric and oligomeric composition inunfermented cacao consists exclusively of (−)-epicatechin and(+)-catechin. (J. Agric. and Food Chem. 51 (2003) 7513). In agreementwith the chromatographic separation described herein, Gu et al. isolateda procyanidin polymer—after the decamer peak—consisting of procyanidinswith an average DP of 14. (Id.). FIG. 3 shows similar separation ofmonomers and oligomers, on the basis of DP, from a Cinnarnomum loureiriiacetone buffer extract, using the improved diol stationary phase andbinary mobile phase process.

Comparison with current methodology. To assess the chromatographicperformance of the Develosil Diol phase as compared to LichrosphereSilica, each column was evaluated under the chromatographic conditionsdescribed by Adamson et al (J. Agric. and Food Chem. 47 (1999) 4184).New chromatographic conditions described for the diol phase (i.e., diolfor the stationary phase, acetonitrile in place of methylene chloride inthe binary mobile phase) did not effect separation of procyanidins whentested in silica. Typical chromatograms from Lichrosphere Silica andDevelosil Diol are exhibited in FIG. 4. The chromatograms demonstrate asignificant difference in retention characteristics under identicalchromatographic conditions. The bonded diol phase only showed similarretention characteristics for the procyanidin monomers, (−)-epicatechinand (+)-catechin. It exhibited stronger retention characteristics fordimer through decamer with retention increasing with degree ofpolymerization. The retention time for the decamer fraction was about50% longer than that observed for Lichrosphere silica under identicalconditions. Consistent with the stronger retention characteristics ofdiol, an increase in speciation was also observed. This was apparentthroughout the entire chromatogram and even impacted the monomer regionat 10 minutes. The diol phase yielded almost baseline resolution of(−)-epicatechin and (+)-catechin while silica showed significantco-elution. This enhanced speciation was not a surprising consequencefor the more retentive bonded phase. However, comparison of peak areasbetween diol and silica stationary phases yielded an unexpected result.The overall peak areas for DP=2 through DP=10 increased on the diolphase as compared to silica. The magnitude of the increases becamegreater with increasing molecular weight. These observations,illustrated by the graph in FIG. 5, are most likely a result of areduction in the adsorption phenomenon. The observations were especiallystriking considering that longer retention typically generates broaderpeak shapes with smaller area.

Preparative scale. Increasing the scale of the chromatography is ofinterest not only for generating standards for further analyticaladvancements but also for elucidating the various physiologicalmechanisms that these phenolic molecules are purported to utilize bygenerating “clean” oligomeric fractions for subsequent biologicalinvestigations. Although the most current analytical processes for theseparation of procyanidins, according to DP, work well and have beenemployed on a routine basis (see, e.g., Rigaud et al., Chromatogr. 654(1993) 179; Hammerstone et al., J. Agric. and Food Chem. 47 (1999) 490;Gu et al., J. Agric. and Food Chem. 50 (2002) 4852), the composition ofthe mobile phase (CH₂Cl₂, CH₃OH, HOAc) severely limits the scale up ofthis process.

One of the major benefits of the diol stationary phase process describedherein is the ability to scale up the process with a smaller concern forsafety issues and disposal costs. Due to solvent choice and the relativesimplicity of the binary mobile phase gradient used for the analyticalscale, the process was able to be transferred to a semi-preparative(250×21.5 mm) and then to the preparative (300×50 mm) scale HPLC systemwith alterations in only the gradient and flow-rate.

A separation of the procyanidin oligomers was achieved with apreparative Diol-Develosil column (300×50 mm) using a mobile phaseconsisting of acetonitrile, methanol and acetic acid. The absorbance ofthe eluate was monitored at 280 nm. Nine peaks were observed in a70-minute run. FIG. 6 shows the trace obtained from the recorder. There,T and C refer to the xanthines, theobromine and caffeine, respectively.The numbered peaks refer to DP. The solvents are low boiling and easy toremove, assisting in the isolation of the oligomers. In addition, theacid content is less.

This methodology is in sharp contrast with that of Adamson et al. (J.Agric. and Food Chem. 47 (1999) 4184) which describes the fractionationof cocoa procyanidin oligomers over a silica column using a gradient ofmethylene chloride-methanol-acetic acid-water. In a single run (180minutes), using a 500×20 mm Supelcosil LC-Si column, Adamson et al. usesnearly 4.5 liters of methylene chloride as part of the mobile phase.Additionally, multiple extraction runs were needed (in Adamson et al.)in order to isolate the oligomeric fractions used as referencematerials.

Comparison to Sephadex. Although the theoretical upper limit of materialthat can be loaded on the preparative diol column is smaller than thatwhich can be loaded onto a 600×100 cm Sephadex LH-50 column (˜4 g versus25 g CP respectively), there are significant improvements with respectto solvent use, time (70 minute run for diol versus several days forSephadex) and resolution. In addition, detection and monitoring duringcollection was possible with the preparative diol system allowing for‘cleaner’ fractions as demonstrated in FIG. 7. Currently, it is thesolubility of the CP extract in the mobile phase that limits size of theamount of material in one injection (2 mL injection loop volume). Thesample preparation involves dissolution of the CP extract in mobilephase (A:B, 25:75 v/v). Under these conditions, a white precipitate(identified as a mixture of xanthines) and a resinous material formed.Increasing the ratio of solvent to solid did not alter these results.After centrifugation, the supernatant was isolated and injected onto thecolumn. Addition of ethanol dissolved the resinous material. Analysis ofthis dissolved material, on the analytical scale process, gave fractionswith DP up to 14. However, injection of the ethanol soluble materialonto the column with the mobile phase that induced precipitation, wasnot desirable for large-scale preparative work. Various methodologiesfor sample preparation (pre-purification via extractions) to increaseinjection load per injection volume, thereby enhancing efficiency, arecurrently being investigated.

Analysis of the isolated peaks (HPLC/FLD) assisted with the assessingthe purity of the fractions, all of which showed purity greater than95%. FIG. 7 shows FLD traces of the individual fractions collected froma preparative HPLC separation of CP extract for oligomeric fractionsDP=1 through DP=7. MS-electrospray ionization in the negative ion modewas employed for the molecular mass assignments of the individualoligomeric fractions. These data are consistent with previous literaturereports (Hammerstone et al., J. Agric. and Food Chem. 47 (1999) 490).The labels in FIG. 6 (i.e. 1-7) correspond to the DP of the oligomericfractions. The first two peaks, labeled T and C were identified as thexanthines, theobromine (T) and caffeine (C), using LC-MS ESI in thepositive ion mode.

Consistent with the stronger retention characteristics of diol, anincrease in speciation was also observed. This was apparent throughoutthe entire chromatogram and even impacted the monomer region at 10minutes. The diol phase yielded almost baseline resolution of(−)-epicatechin and (+)-catechin while silica showed significantco-elution.

Comparison of peak areas between diol and silica stationary phases,yielded an unexpected result: the overall peak areas for DP=2 throughDP=10 increased on the diol phase as compared to the silica phase. Themagnitude of increase became greater with increasing molecular weight(i.e., DP), as illustrated in FIG. 5. The observations were especiallystriking considering that longer retention typically generates broaderpeak shapes with smaller area.

It will be understood by one of ordinary skill in the art that, althoughthe above examples use diol as a stationary phase, other polar bondedstationary chromatographic phases may be used to obtain the sameresults. Suitable stationary phases, in addition to a diol phase,include but are not limited to, a glycerol phase, an amino (preferably,propyl amine) phase, a cyano (preferably, cyanopropyl) phase, atrimethylsilyl phase, a dimethylsilyl phase, a propyl phase, a butylphase, a pentyl phase, a hexyl phase, a phenyl phase, a halogenatedphase and a nitro phase. Also, while the disclosed particle size is 5μm, it will be understood by those of ordinary skill in the art that,particularly with respect to a diol stationary phase, any commerciallyavailable particle size may be used for the separation. Presently,commercially available diol particle sizes include 3, 5 and 10 μm.

Similarly, although the above examples use a binary mobile phase ofacetonitrile and methanol, the binary mobile phase may be a combinationof any polar aprotic solvent (A phase) followed by any polar proticsolvent (B phase). Suitable polar aprotic solvents for the (A) phaseinclude, in addition to acetonitrile, acetone, cyclohexanone, methylethyl ketone, methyl tert-butyl ether, diethyl ether, dimethyl ether,methyl acetate, ethyl acetate and nitromethane. Suitable polar proticsolvents for the (B) phase include, in addition to methanol, ethanol,n-propanol, isopropanol, n-butanol and isobutanol. It will be understoodthat the proportion of polar aprotic solvent in the A phase and polarprotic solvent in the B phase may be between 0 and 100 percent byvolume, preferably is greater than 50% by volume, and even morepreferably is greater than 90% by volume. The remainder of the A phaseand B phase may be any mineral or organic acid (i.e., acetic acid)and/or water.

Accordingly, the improved processes and system of the present inventionfor separating polar protic oligomers based on degree of polymerizationon an analytical scale and for separating and isolating the oligomers ona preparative scale provide several advantages over existing approaches.The polar bonded stationary chromatographic phase is more robust thansilica, and can tolerate a wider range of solvents. The long termadsorption properties of silica are compromised in the presence ofwater, whereas with the polar bonded stationary chromatographic phase,such as a diol or glycerol stationary phase, this is not a concern. Whendirectly compared to silica, the diol phase was shown to reduce oreliminate surface adsorption, thereby providing for increased speciationof individual oligomers, including especially oligomers with DP of atleast 10. A binary mobile phase is used, rather than the typicaltertiary or quaternary system, thereby making the process readilyadaptable to researchers lacking sophisticated quaternary HPLC pumps.The solvents used in the disclosed process are less dangerous and moreenvironmentally friendly than those currently employed, vis., thecarcinogen/mutagen methylene chloride and the CNS depressanttetrahydrofuran (THF). Finally, the process allows ‘clean’ physicalisolation of fractions according to DP which can be advantageous withrespect to generating standards for further research or for purificationof individual oligomers.

In addition to separating polar protic oligomers on the basis of DP, amodification of the mobile phase gradient used to separate theprocyanidin oligomers (according to DP) has been found to enablegeneration of a decaffeinated and detheobrominated cocoa polyphenolextract. With respect to recovery: starting with 1 g (40% CP—i.e. 400 mgof material) 134 mg of oligomers (DP>2) were recovered. The mass ofrecovered monomer was 39 mg.

Separation of xanthines (caffeine and theobromine) from cocoapolyphenols was carried out on an Agilent 1100 HPLC system using aDevelosil Diol 300×50 mm, 100 μm column (stationary phase), at roomtemperature (i.e., ˜25° C.). The composition of the binary mobile phasewas as follows: the (A) phase comprised acetonitrile:acetic acid 99:1(v/v) and the (B) phase comprised methanol:water:acetic acid 95:4:1(v/v/v). The flow rate for semi-preparative work was set at 30 ml/min.Separations were effected by a linear gradient as follows: 0 minutes, 0%B; 15 minutes, 0% B; 20 minutes, 100% B; 30 minutes, 100% B. Analysiswas by UV detection at 280 nm, and fluorescence.

FIG. 8 shows an LC trace generated by the preparative HPLC system. Peak1 is theobromine, peak 2 is caffeine, peak three is the monomer(flavanol) and peak 4 is oligomers with DP>1. Holding initial mobilephase conditions for 15 minutes (i.e., 0% B phase, 100% A phase),theobromine, caffeine and monomer were eluted first. Adjusting themobile phase to high polarity allowed for quick recovery of CP oligomers(DP>1).

FIG. 9 displays the analyses of the fractions. Trace (a) is thefluorescence detection of the CP extract. Trace (b) is the UV detectionof the same material. It is known that xanthines do not fluorescesubstantially and CPs do not display strong signals under UV detection.Using both detection modes thus gives a more comprehensive picture.

Trace (c) shows the UV trace of the xanthines removed from the CPextract. Although the analysis of individual fractions of theobromineand caffeine are not displayed, they can be separated and physicallyisolated. Additionally, the monomer can be separated and isolated fromthe xanthines. See trace (d).

Trace (e) shows the UV trace of the CP extract after removal of themonomer and xanthines. As is evident, the sample contains only oligomersof DP>2.

While the invention has been described with respect to certain specificembodiments, it will be appreciated that many modifications and changesmay be made by those skilled in the art without departing from theinvention. It is intended, therefore, by the appended claims to coverall such modifications and changes as may fall within the true spiritand scope of the invention.

1-40. (canceled)
 41. A normal phase high performance liquidchromatographic process for separating, eluting, and isolatingindividual polar protic monomer(s) and/or polar protic oligomer(s)comprises the steps of: (a) introducing a liquid sample containing thepolar protic monomer(s) and/or the polar protic oligomer(s) into aliquid chromatography column packed with a polar bonded stationary phaseselected from the group consisting of a diol phase, a glycerol phase, anamino phase, a cyano phase, a trimethylsilyl phase, a dimethylsilylphase, a halogenated phase, and a nitro phase; (b) passing a binarymobile phase comprising an A phase consisting essentially of a polaraprotic solvent and a B phase consisting essentially of a polar proticsolvent through the packed column to elute the individual polar proticmonomer(s) and/or the individual polar protic oligomer(s) on the basisof degree of polymerization; (c) isolating one or more individualfractions containing the polar protic monomer(s) and/or the polar proticoligomer(s); and (d) optionally recovering the isolated polar proticmonomer(s) and/or the isolated polar protic oligomer(s).
 42. The processof claim 41, wherein the polar protic oligomer(s) are proanthocyanidins,hydrolyzable tannins, oligosaccharides, oligonucleotides, peptides,acrylamides, polysorbates, polyketides, poloxamers, polyethyleneglycols, polyethylene alcohols, or polyvinyl alcohols.
 52. The processof claim 51, wherein the proportion of polar aprotic solvent in the Aphase is greater than 50% by volume; wherein the proportion of the polarprotic solvent in the B phase is greater than 50% by volume; and whereinthe remainder of the A phase and the remainder of the B phase is anymineral acid, organic acid, and/or water.
 53. The process of claim 52,wherein the proportion of the polar aprotic solvent in the A phase isgreater than 90% by volume; wherein the proportion of the polar proticsolvent in the B phase is greater than 90% by volume; and wherein theorganic acid is acetic acid.
 54. The process of claim 52, wherein the Aphase consists essentially of a mixture of about 98% acetonitrile andabout 2% acetic acid (v/v).
 55. The process of claim 52, wherein the Aphase consists essentially of a mixture of about 99% acetonitrile and 1%acetic acid (v/v).
 56. The process of claim 52, wherein the B phaseconsists essentially of a mixture of about 95% methanol, about 3% water,and about 2% acetic acid (v/v/v).
 57. The process of claim 52, whereinthe B phase consists essentially of a mixture of about 95% methanol,about 4% water, and about 1% acetic acid (v/v/v).
 58. The process ofclaim 41, wherein the stationary phase has a particle size from about 3μm to about 10 μm.